Monomer protein with bone morphogenetic activity and medicinal agent containing the same for preventing and treating diseases of cartilage and bone

ABSTRACT

This invention provides for a protein of the TGF-β superfamily in which the cysteine involved in the normal formation of homodimers is changed to another amino acid. These mutant proteins, as monomers, display higher bone morphogenetic activity than the wild-type protein dimers. Also provided is a method for producing and isolating these monomers by plasmid driven expression in various host systems including  E. coli . In addition, the invention discloses the use of an agent containing purified monomers in preventing and treating diseases and problems affecting bone and/or cartilage.

CROSS REFERENCE TO RELATED APPLICATION

This application is a Divisional of application Ser. No. 10/751,451which issued as U.S. Pat. No. 7,235,241 on Jan. 6, 2004, which is adivisional of application Ser. No. 09/701,121 filed on Jan. 3, 2001, nowabandoned, which is a 35 U.S.C. 371 National Phase Entry Applicationfrom PCT/IB99/00866, filed May 14, 1999, which claims the benefit ofJapanese Patent Application No. 10/141,379 filed on May 22, 1998, thedisclosure of which is incorporated herein in its entirety by reference.

BACKGROUND OF THE INVENTION

(1) Field of the Invention

The present invention relates to a monomer protein having an amino acidsequence belonging to TGF-β superfamily, of which cysteine related to adimer formation of a protein has been replaced with another amino acid.Moreover, the present invention relates to a method for preparing saidmonomer protein in a large amount and with a high purity by usingEscherichia coli transformed with a plasmid containing a DNA sequencethat can express said monomer protein. Furthermore, the presentinvention relates to an agent containing said monomer protein forpreventing and treating a disease affecting bone and/or cartilage.

(2) Description of the Related Art

Currently, there are known estrogen, calcitonin, vitamin D3, itsderivatives and derivatives of bisphosphonic acid as preventive ortherapeutic agents for bone diseases. Recently, it has been reportedthat a bone morphogenetic activity is found in a series of a bonemorphogenetic protein (hereinafter referred to as “BMP”) belonging toTGF-β superfamily, from BMP-2 to BMP-14.

Moreover, it has been reported that a protein named GDF-5 or human MP52has a bone morphogenetic activity (WO93/16099, WO95/04819, WO94/15949and Nature Vol. 368, 1994, p. 639-643). It is considered that maturehuman MP52 is a protein having 120 amino acid residues starting withalanine at an N-terminal, and its amino acid sequence has been describedin these patent applications.

These proteins exist as a homodimer having a single disulfide bond innature. On the contrary, the manufacture of their recombinant protein iscarried out using their homodimers or heterodimers to yield a proteinshowing the activity. For example, human MP52 has been reported in thepublication of unexamined application, JP 031098/97.

Meanwhile, there are two types named type I receptor and type IIreceptor in the receptors of TGF-β superfamily. Intercellular signaltransmission via receptors of TGF-β superfamily containing these bonemorphogenetic proteins (dimers) requires simultaneous combination ofthese proteins to both type I and type II receptors, and it isconsidered that a polymer is formed by gathering of two or more dimersto do intercellular signal transmission (Bone, Vol. 19, 1996, p.569-574). It has been considered that for polymer formation it isimportant that the protein should be a dimer. The activity in a monomerhas not yet been found. Moreover, preparation for these monomerrecombinants has not yet been carried out.

SUMMARY OF THE INVENTION

The present inventors have attempted a mass production of human MP52monomers by a genetic engineering technology using Escherichia coli.Namely, the present inventors constructed a plasmid of DNA sequenceencoding the amino acid sequence having 119 residues described in SEQ IDNO: 1 of the Sequence Listing, among which the codon of the cysteineresidue of No. 83, that is related to a disulfide bond between MP52monomer molecules, was converted to the codon of alanine. In addition,the inventors have succeeded in expressing a large amount of human MP52monomers using Escherichia coli by using the plasmid and refolding toproduce monomers of the protein described in SEQ ID NO: 1 of theSequence Listing with a high purity and a very high yield.

It has been surprisingly found that the monomer has the activity toinduce differentiation to osteocytes in some cell lines (MC3T3-E1 andATDC5) despite that in conventional understanding, only a dimer has abone morphogenetic activity. The present invention has been completed byobserving that the activity to induce differentiation is two-fold higherthan that of the dimer on the basis of weight concentration.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a plasmid map of the expression vector (pKOT279) obtained inExample 1 (2).

FIG. 2 is a comparative figure of osteoblast differentiation promotingactivities between the monomer of the present invention and human MP52dimer. (A) shows the activity in MC3T3-E1 cells and (B) shows that inATDC5 cells. The white circle shows the activity of the monomer and theblack circle shows that of human MP52 dimer.

DESCRIPTION OF THE PREFERRED EMBODIMENT

Namely, the present invention relates to a monomer protein having anamino acid sequence belonging to TGF-β superfamily, of which cysteinerelated to a dimer formation of the protein has been replaced withanother amino acid, a method for expressing said monomer protein, and anagent for preventing and treating a disease affecting bone and/orcartilage containing one or more than one said monomer proteins.

The present invention relates to a monomer protein having an amino acidsequence belonging to TGF-β superfamily, of which cysteine related to adimer formation of the protein has been replaced with another aminoacid. The TGF-β superfamily of the present invention means BMP-2, BMP-4,BMP-5, BMP-6, BMP-7, BMP-12, BMP-13, BMP-14, human MP52, GDF-5, GDF-6,GDF-7, etc. Another amino acid may be any amino acid selected from agroup consisting of alanine, threonine, serine and valine inconsideration of the size of an amino acid side chain. The mostpreferable amino acid is alanine.

The present invention relates to a monomer protein having an amino acidsequence described in SEQ ID NO.: 1 of the Sequence Listing. In detail,the monomer protein is a protein in which cysteine is replaced withalanine, and the aforesaid cysteine contributes to intermoleculardisulfide bond of a human MP52 dimer having an intermolecular disulfidebond, and is present at the 83^(rd) position of the amino acid sequenceof SEQ ID NO.: 1 of the Sequence Listing. The monomer protein obtainedby the present invention shows a two-fold higher activity in inducingdifferentiation than a dimer protein made from the monomer protein.

Furthermore, the present invention relates to a method for preparationof said monomer protein to express by using Escherichia coli, yeast,insect cells, and mammal cells that have been transformed by a plasmidhaving a DNA sequence capable of expression of said monomer protein. Indetail, the present invention relates to a method for preparation of aprotein having 119 amino acid residues derived from human MP52represented by SEQ ID NO.: 2 of the Sequence Listing, by employingEscherichia coli. In other words, the present invention relates toconstruction of a plasmid having a DNA sequence that encodes an aminoacid sequence in which methionine is added to the N-terminal of theamino acid sequence derived from human MP52 in which alanine hasreplaced cysteine of the 83^(rd) position from 119 residues representedby SEQ ID NO.: 1 of the Sequence Listing. For human MPS2 cDNA, a matureportion was solely amplified by polymerase chain reaction (PCR method)by using a plasmid vector as a template DNA containing cDNA described inWO93/16099. The PCR method used in the invention means generalamplification from a very small amount of a fragment of DNA or RNA of anucleic acid by the method described in U.S. Pat. No. 4,683,195.

In the present invention, a mutant monomer protein was obtained byconstruction of a plasmid having a DNA sequence that encodes an aminoacid sequence in which methionine is added to the N-terminal of theamino acid sequence represented by SEQ ID NO.: 1 of the SequenceListing, by transformation of the plasmid to Escherichia coli, bysolubilization of the inclusion body obtained by culturing theEscherichia coli and by purification. The present invention relates to amethod for preparation of the protein by refolding to have an activityand purifying said protein to a monomer protein described in SEQ ID NO.:2 of the Sequence Listing. Concretely, for the monomer protein of thepresent invention, MP52 mutant monomer protein was obtained by applyingthe solubilized inclusion bodies of Escherichia coli to a SP-SepharoseFF column (Amersham Pharmacia Biotech) and to Superdex 200 pg column(Amersham Pharmacia Biotech). Subsequently, the purified monomer proteinof the present invention is obtained by refolding and then by passingthrough a reversed phase HPLC RESOURCE RPC column (Amersham PharmaciaBiotech). The physical and chemical properties of the present monomerprotein obtained are analyzed on the basis of data of an N-terminalamino acid sequence, an amino acid composition, and electrophoresis.

The biological properties of the monomer protein of the presentinvention were evaluated by the activity to induce differentiation oftwo kinds of osteoblast cell lines of which promoting alkalinephosphatase activity was already found in a human MP52 dimer. Incomparison in the weight concentration, the monomer protein of thepresent invention showed a two-fold higher activity than that of theconventional dimer protein.

The present invention relates to a preventive or therapeutic agent forcartilage and/or bone diseases having amino acid sequence represented bySEQ ID NO.: 2 of the Sequence Listing as an effective ingredient. Indetail, the monomer protein of the present invention has an activity toinduce differentiation, i.e., an morphogenetic activity for cartilageand bone, and therefore, relates to a preventive or therapeutic agentfor osteoporosis, congenital bone and/or cartilage diseases, andosteoarthritis such as joint osteoarthritis and hip jointosteoarthritis, or arthrosteitis, damage of cartilage such as damage ofmeniscus, regeneration of bone and cartilage deficit caused by injuryand tumor dissection, bone and cartilage deficit, fracture, congenitalcartilage and/or bone diseases such as achondroplasia,dyschondrogenesis, achondrogenesis, palatoschisis, and dysosteogenesis,and a deficit of root of teeth and a tooth socket.

Furthermore, the protein of the present invention, having bone andcartilage morphogenetic activity, can be used for therapy of bone graftin an aesthetic surgery field. The therapy includes a field ofveterinary surgery.

As in systemic administration method, intravenous, intramuscular, andintra-abdominal administrations are possible; in an intravenousadministration, an intravenous drip can be applied in addition to ageneral intravenous injection.

An injection preparation can be, for example, a powder preparation forinjection. In the case, one or more kinds of appropriate water-solubleexcipient such as mannitol, sucrose, lactose, maltose, glucose, orfructose are added to dissolve in water, divided into vials or ampoules,freeze-dried, and hermetically sealed to make as a product.

For a local administration method, there is a method to cover thesurface of a cartilage, bone, or tooth of the site with the presentprotein by using collagen paste, fibrin glue, or other adhesives. Amongthem, a bone used for bone graft can be also applied to an artificialbone conventionally used as well as a natural bone. The artificial bonesinclude bones made of natural materials or artificial inorganicmaterials such as metals, ceramics, and glasses. The artificialinorganic materials are preferably exemplified by hydroxyapatite. Forexample, a metal is used for an internal material and hydroxyapatite foran external material of an artificial bone. Furthermore, the presentprotein can be administered to a carcinomatous tissue to enhancereconstruction of a bone. It is also possible to use for cartilagegrafting.

An administrative dose is determined by a physician in charge inconsideration of the following various factors affecting the action ofthe present protein: the weight of bone and cartilage to reconstruct,the site and condition of the damage of bone and cartilage, sex and ageof a patient, severity of the infection, administration duration, andother clinical factors. The dose can vary according to the kind of acarrier used for reconstruction that is realized in combination with thepresent protein. In general, concerning the dose, ca. 10-10⁶ ng as thepresent monomer protein for a given wet weight of a bone and cartilagein the use as a composition containing a carrier and 0.1-10⁴ μg for onepatient as an injection for local and in systemic application arepreferably administered in the frequency ranging from once a day to oncea week.

A multiplier effect can be expected by simultaneous application of aknown growth factor such as insulin-like growth factor-I forregeneration of a bone and cartilage.

Thus, a monomer made by substitution of cysteine of a protein belongingto TGF-β superfamily and industrial manufacture for a monomer have notbeen reported. The monomer has a morphogenetic activity for cartilageand bone and is useful as a therapeutic agent for cartilage and/or bonediseases. Furthermore, the monomer protein of the present inventionshows a two-fold higher activity per weight than that of a dimer of theprotein and allows a half reduction of an effective dose of atherapeutic agent for cartilage and/or bone diseases. This fact can beapplied to manufacture of before-mentioned bone morphogenetic factorsbelonging to TGF-β superfamily.

The monomer protein derived from human MP52 and having an amino acidsequence described in SEQ ID NO.: 2 of the Sequence Listing has atwo-fold higher activity in a osteoblast cell line to inducedifferentiation than that of the dimer and useful as a preventive ortherapeutic agent for cartilage and/or bone diseases. Furthermore, achange of an amino acid of the monomer protein of the present inventionreduces cysteine and thus, it makes easy preparation of a mass and puremonomer protein possible by using Escherichia coli.

EXAMPLES

This invention shall be more illustratively explained by way of thefollowing Examples. The following Examples are to be considered in allrespects as illustrative and not restrictive.

Example 1 Preparation of a Human MP52 Monomer Expression Vector

(1) Isolation of a Mature Region of a Human MP52 Mutant

The human MP52 monomer was prepared by replacing cysteine residue whichis regarded as forming a dimer with another amino acid residue in orderto prevent the formation of a dimer with the human MP52 monomer. In thepresent invention, the codon of cysteine (TGC) of the 83^(rd) of themature human MPS2 starting with proline described in SEQ ID NO.: 1 ofthe Sequence Listing of WO 96/33215 was converted to the codon ofalanine (GCC).

The substitution of an amino acid residue was carried out by using a PCRprimer (forward direction) in which an objective mutation has beenintroduced with reference to the mutation method (Section 8.5) bypolymerase chain reaction (PCR) described in Current Protocols inMolecular Biology (John Wiley & Sons, Inc.). The sequence of the PCRprimer used was described in SEQ ID NO.: 3 as a sense primer and in SEQID NO.: 4 as a reverse primer.

PCR was performed by using a human MP52 expression vector (pKOT245).described in WO96/33215 as a template DNA (10 ng), each 10 μM senseprimers and reverse primers, dNTP of 0.4 mM, MgCl₂ of 2.5 mM, and LA TaqDNA polymerase (5U, Takara Shuzo Co., Ltd; catalog No. RRO13A) in thesame test tube. The 30 cycles of reaction was operated of which onecycle included denaturation (94° C., 1 min), primer annealing (55° C., 1min), and primer elongation (72° C., 2 min). The PCR product wasdigested by restriction enzymes NcoI and HindIII, separated byelectrophoresis with 1.5% low melting point agarose (FMC BioProductsCo., catalog No. 5170B) and purified to obtain a DNA fragment having aca. 170 bases as an objective product.

The human monomer MP52 expression vector (pKOT279) was prepared byreplacing a DNA fragment of NcoI-HindIII in which mutation wasintroduced by aforementioned method with NcoI-HindIII region of a humanmonomer MP52 expression vector (pKOT277) made by modifying a humanmonomer MP52 expression vector (pKOT245) described in WO96/33215.Concretely, by preparing the human monomer MP52 expression vector(pKOT277) from which lacZ promoter, that is transcribed in the reversedirection to a MP52 existing in the downstream of the terminator of thehuman monomer MP52 expression vector (pKOT245) described in WO96/33215,by digesting said MP52 expression vector (pKOT277) by restrictionenzymes NcoI and HindIII, separating by electrophoresis in 1.5 a lowmelting point agarose (FMC BioProducts Co., cat. No. 5170B) and bypurifying, a DNA fragment having 2717 base pairs was obtained for anobjective product. The DNA fragment and the DNA fragment of ca. 170 basepairs to which mutation was introduced, were ligated by using DNALigation Kit (Takara Shuzo Co., Ltd., catalog No. 6021) to prepare ahuman monomer MPS2 expression vector (pKOT279, 2.9 kb). The vector wasdeposited in National Institute of Bioscience and Human-Technology,Agency of Industrial Science and Technology, Ministry of InternationalTrade and Industry, 1-3, Higashi 1-chome, Tsukuba-shi Ibaraki-ken305-8566 Japan, in Feb. 5, 1998 (Deposit no. Bikoukenki no. FERMP-16625) and transferred to the International Depository Authority underBudapest Treaty on Feb. 3, 1999 (Deposit No. FERM BP-6637). For the basesequence of the human MP52 monomer expression vector of the presentinvention, introduction of the objective mutation and correctness of thebase sequence (other sequence than that of the site to which a mutationwas introduced) of the human MP52 produced were confirmed by using a DNAsequencer (Amersham Pharmacia Biotech, ALF).

(2) Transformation

Transformation was experimented according to rubidium chloride method ofKushner et al. (Genetic Engineering p. 17, Elsevier. 1978). Namely,pKOT279 was introduced to Escherichia coli W3110M according to abovemethod to make the Escherichia coli to express a protein in the presentinvention.

Example 2 Cultivation

(1) Cultivation

The Escherichia coli to express a protein of the present invention wasprecultured in a modified SOC culture medium (Bacto tryptone 20 g/L,Bacto yeast extract 5 g/L, NaCl 0.5 g/L, MgCl₂ 0.95 g/L, and glucose 3.6g/L), 100 mL of cell suspension (Bacto tryptone 20 g/L, citric acid 4.3g/L, K₂HPO₄ 4.675 g/L, KH₂PO₄ 1.275 g/L, NaCl 0.865 g/L, FeSO₄.7H₂O 100mg/L, CuSO₄.5H₂O 1 mg/L, MnSO₄.nH₂O 0.5 mg/L, CaCl₂.2H₂O 2 mg/L,Na₂B₄O₇.10H₂O 0.225 mg/L, (NH₄)₆Mo₇O₂₄ 0.1 mg/L, ZnSO₄.7H₂O 2.25 mg/L,CoCl₂.6H₂O 6 mg/L, MgSO₄.7H₂O 2.2 g/L, thiamine HCl 5.0 mg/L, methionine2 g/L, and glucose 3 g/L) was added to 5 L of a culture medium forproduction to culture in a 10 L culture vessel with aerated stirring,isopropyl-β-D-thiogalactopyranoside of 1 mM concentration in a stagereached a logarithmic multiplication prophase (OD₅₅₀=50) was added toculture by OD₅₅₀ beyond 150. In the culture, the temperature wasregulated to 31° C. and the pH was regulated to 7.2 by adding ammonia.Dissolved oxygen concentration was regulated to 50% of air saturation byincreasing stirring speed in order to prevent decrease in dissolvedoxygen concentration. A 50% glucose solution containing 0.1 M phosphatewas added to make glucose concentration 0.2% with reference to rapidrise of dissolved oxygen concentration as an indication in order to makea higher cell concentration in culture.

(2) Preparation of the Inclusion Bodies from Escherichia coli

The culture solution obtained by said method was passed three timesthrough a high pressure homogenizer (LAB40-10RBF1, APV, Gohrin Co.)under 560 bar pressure to break cells and centrifuge to collect aprecipitate containing the inclusion bodies.

Example 3 Purification

(1) Solubilization of the inclusion bodies from Escherichia coli

The inclusion bodies collected were washed twice with 20 mM Tris-HClbuffer solution (pH 8.3) containing 1 M urea and 5 mM EDTA andcentrifuged at 4° C. and 3,000×g for 30 min; the precipitate obtainedwas solubilized by sonication in 20 mM Tris-HCl buffer solution (pH 8.3)containing 8 M urea, 50 mM NaCl, 64 mM DTT, and 5 mM EDTA.

(2) Purification of denatured monomer protein The solubilized solutionwas centrifuged at 4° C. and 20,000×g for 30 min and the supernatant wascollected. The supernatant collected was applied to SP-Sepharose FF(Amersham Pharmacia Biotech) column equilibrated with 20 mM Tris-HClbuffer solution (pH 8.3), 6 M urea, 10 mM DTT, and 1 mM EDTA, washedwith the solution, and eluted with the solution containing 0.4 M NaCl.The eluate was subjected to gel filtration with a Superdex 200 pg column(Amersham Pharmacia Biotech) equilibrated by 20 mM Tris-HCl buffersolution (pH 8.3), 6 M urea, 0.5 M NaCl, 10 mM DTT, and 1 mM EDTA toobtain a single denatured monomer protein.

(3) Refolding

50 mM Na-Glycine buffer solution (pH 9.8), 0.5 M NaCl, 20 mM CHAPS, and3 mM GSSG (oxidized glutathione) of nine times quantity were added tothe solution of the denatured monomer protein obtained by abovetreatment followed by stirring to refold at 4° C. for 20 h.

(4) Purification of a Monomer Protein Having an Activity.

The sample refolded was diluted 2.8-times with 14 mM NaH₂PO₄ andsubjected to isoelectric precipitation. The precipitate was collected bycentrifugation at 3,000×g for 20 min and dissolved in 0.05% TFA. Thesolution was applied to a RESOURSE RPC column (Amersham PharmaciaBiotech) of reverse-phase HPLC previously equilibrated with 0.05% TFAand eluted with 0.05% TFA and 0-50% acetonitrile gradient. The eluatewas monitored by an absorptiometer at 280 nm absorbancy to obtain afraction of purified monomer protein of the present invention. To theprotein fraction, 5 N NaOH was added to make in the range of between pH6.5 and 7.5 for precipitation in isoelectric point. The precipitate wascollected by centrifugation of 10,000×g for 10 h and dissolved in 10 mMHCl to make ca. 3 mg/mL to obtain a monomer protein having an activityof the present invention.

(i) N-Terminal Sequence Analysis

The N-terminal analysis of the amino acid composition of the purifiedmonomer protein of the present invention obtained above was carried outby using a sequencer (Applied Biosystem, Model 476A).

(ii) Amino Acid Composition Analysis

The amino acid composition of the purified monomer protein of thepresent invention obtained above was examined by an amino acid analyzer(Waters, PICO. TAG. WORK STATION).

(iii) Electrophoretic Analysis

The molecular weight of the purified monomer protein of the presentinvention obtained above was investigated by SDS-PAGE under anon-reduced condition to be a molecular weight of ca. 1.4 kDa.

As the results given by (i), (ii), and (iii), it has been found that themonomer protein of the present invention is a monomer protein having 119amino acid residues of which N-terminal starts with Pro shown in SEQ IDNO: 2 of the Sequence Listing.

Example 4 Measurement of Biological Activity

A differentiation inducing activity was evaluated by employing twocultured cell lines; ATDC5 (Riken Gene Bank, RCB 0565) to differentiatelike a cartilage cell derived from a mouse embryonic cell and MC3T3-E1(Riken Gene Bank, RCB 1126) having properties like those of anosteoblast derived from a mouse, on the basis of reference to alkalinephosphatase promoting activity of said protein. The result is shown inFIG. 2.

ATDC5 and MC3T3-E1 of the concentration of 10,000 cells per 1 mL weresuspended in DF culture medium (Gibco Ltd.) containing 5% bovine fetusserum and in MEM-α⁻ medium (Gibco Ltd.) containing 10% bovine fetusserum, respectively, and inoculated in 24 plates at 1 mL per 1 well toculture at 37° C. for 3 days under 5% CO₂.

Subsequently, the cells were rinsed with the MEM-α− medium withoutserum, a natural dimer or a monomer protein diluted gradationally withthe MEM-α⁻ medium containing 0.3% bovine albumin was added 0.5 mL per 1well to start induction of differentiation. The cultivation was carriedout for 3 days, the cells were rinsed with PBS (20 mM phosphate buffersolution, 150 mM NaCl, pH 7.4) twice and 250 μL of cytolytic solution(0.2% NP-40, 1 mM MgCl₂) was added and kept standing at 37° C. for 2hours. Following this step, the total volume of the cytolytic solutioncontaining cells broken was transferred to a micro tube and centrifuged(10,000×g, 5 min) to use its supernatant for assay.

An enzyme activity was measured by observing the rise of absorbancy ofp-nitrophenol (pNp) being the dissociated product derived fromp-nitrophenyl phosphate as the substrate of the final concentration of10 mM by dissolving in 0.1 M glycine buffer, pH 10.4, 1 mM ZuCl₂, and 1mM MgCl₂, at 405 nm.

The rise of absorbancy was observed every 2 min for 40 min and thealkaline phosphatase promoting activity (μM pNp/min) was calculated onthe basis of the data of the range showing linearity.

In addition, the protein concentration of the same supernatant was knownby using a BCA Protein Assay Kit (Amersham Pharmacia Biotech) and thealkaline phosphatase activity per protein was represented by nmolpNp/min/mg protein.

1. A method for inducing the differentiation of osteoblast cells, comprising administering a monomer protein comprising an amino acid sequence belonging to the TGF-β super family, wherein said monomer protein comprises the amino acid sequence described in SEQ ID NO:2 or the amino acid sequence described in SEQ ID NO:2 wherein alanine at position 83 is replaced with a serine, threonine or valine and wherein said monomer protein induces differentiation of osteoblasts measured by promoting alkaline phosphatase activity, to a patient in need of differentiation of osteoblast cells.
 2. The method according to claim 1, wherein said monomer protein is MP52.
 3. The method according to claim 1, wherein said patient is suffering from a condition selected from the group consisting of osteoporosis, osteoarthritis, arthrosteitis, bone fracture, and lack of a tooth root and/or tooth socket.
 4. The method according to claim 1, wherein said monomer protein is administered systemically or locally.
 5. The method according to claim 4, wherein said monomer protein is administered by intravenous, intramuscular or intra-abdominal administration.
 6. The method according to claim 4, wherein said monomer protein is administered by applying a collagen paste, artificial bone, fibrin glue or adhesive comprising the monomer protein.
 7. The method according to claim 1, wherein said patient is suffering from a congenital bone and/or cartilage disease resulting in a bone and/or cartilage deficit.
 8. The method according to claim 7, wherein said patient is suffering from a congenital bone and/or cartilage disease selected from the group consisting of achondroplasia, dyschondrogenesis, achondrogenesis, palatoschisis and dysosteogenesis.
 9. The method according to claim 1, wherein said patient is suffering from cartilage damage.
 10. The method according to claim 9, wherein said patient is suffering from damage to a meniscus.
 11. The method according to claim 4, wherein said monomer protein is administered by injection.
 12. The method according to claim 6, wherein said artificial bone comprises natural materials or artificial inorganic materials.
 13. The method according to claim 12, wherein said artificial inorganic materials are selected from the group consisting of metals, ceramic, glasses and/or hydroxyapatite.
 14. The method according to claim 12, wherein said artificial bone comprises metal for an internal material and hydroxyapatite for an external material.
 15. The method according to claim 1, wherein said patient is in need of a cartilage and/or bone graft. 